In order to protect civilian and military populations, it is critical to improve the diagnosis of NIAID Category A-C priority pathogens. More rapid, more sensitive, more specific, and more informative diagnosis than currently possible will allow earlier and targeted treatment of the individual patient, improved strategic and tactical responses by civilian and military authorities, and more effective remediation activities. The goal of the proposed research is to develop a prototype instrument that will allow the rapid, sensitive, and specific identification NIAID Priority Pathogens in clinical samples by nucleic acid sequencing. The proposed system will identify three pathogens, B. anthracis (vegetative cells and spores), Y. pestis, and B. pseudomallei;additional Category AC Priority Pathogens will be added following successful completion of the proposed two-year program. The proposed system will generate approximately 500 bp of QV20 sequence from each of eight loci per biothreat--a number of loci large enough to provide strain-specific biothreat identification but small enough to allow generation of results within one hour of clinical sample collection. In contrast to other detection technologies, the proposed sequencing system will allow identification of closely related species and strains, genetically engineered biothreats, and emerging agents that are invisible to conventional nucleic acidand immuno-based assays. The system will consist of three components: (1) A Smart Cartridge (SC) will accept a clinical sample (e.g. 5 cc blood), and extract and purify DNA;(2) An Integrated Biochip (IB) will perform highly multiplexed amplification, Sanger sequencing, and electrophoretic separation and laserinduced detection;(3) A ruggedized fully integrated instrument will accept the SC and IB and performs all required process steps. The proposed program is focused on the following activities: (1) Design and testing of biothreat primers and development of analytic software (2) Design, fabrication, and testing of a SC that incorporates Network Biosystem's existing cell separation and microfluidic DNA purification technologies with a spore lysis system to be developed at NetBio;(3) Design, fabrication, and testing of an IB that integrates microfluidic amplification, sequencing, and electrophoretic separation and detection;and (4) Testing of the SC, IB, and instrument using simulated clinical samples and animal models. The proposed two-year program will result in development of a breadboard instrument in Year 1 and a more mature prototype in Year 2. The prototype will allow demonstrations of the system to validate the rapid biothreat sequencing approach. Following program completion, the prototype design will be optimized and finalized for commercial manufacturing.